Regenerative medicine is a broad field in which a multitude of technologies are employed to enhance the body's ability to heal itself. One such technology is cellular therapy. Several cell types have been used for cellular therapy including, but not limited to, embryonic, neonatal, somatic, xenogenic, and induced pluripotent cells. Mesenchymal stem cells (MSCs) in particular have demonstrated therapeutic efficacy by regenerating skeletal tissues such as bone, cartilage, and tendons while also treating neuronal degeneration and supporting hematopoietic engraftment. See, e.g., Doucet et al., J Cell Phys, (2005), 205: 228-236 and Lange et al., J Cell Physiol, (2007), 213: 18-26.
Previous efforts have proliferated donor cells ex vivo using a growth medium containing fetal bovine serum (FBS). The presence of FBS in the culture medium, despite washing the donor cells prior to transfer to the recipient, has led to the generation of antibodies against FBS and has resulted in arthus-like reactions. Selvaggi et al., Blood, (1997), 89(3): 776-9. Concerns regarding the transfer of animal prion, viral, and zoonose contamination also surround the use of FBS. The potential complications associated with using FBS in growth media have generated a particular interest for non-xenogenic growth supplements capable of maintaining the functionality of cells.
Autologous and allogeneic human sera were both tested as a replacement for FBS in efforts to proliferate MSCs ex vivo. Allogeneic serum resulted in cell growth arrest and death of MSCs. Shandadfar et al., Stem Cells, (2005) 23(9): 1357-66. Although autologous serum proliferates MSCs effectively, pragmatic concerns such as cost and availability of serum make this approach somewhat impractical. See, e.g., Kobayashi et al., J Bone Joint Surg Br, (2005), 87(10): 1426-33. Previous investigations indicate that growth factors released from human platelets effectively enhance the growth of several cell types, including MSCs. Consequently, recent efforts have focused upon using platelet lysate in lieu of FBS as a cell culture supplement.
Platelets are capable of releasing multiple growth factors such as multiple platelet-derived growth factors (PDGFs), insulin-like growth factor 1 (IGF-1), and transforming growth factor beta (TGF-β). Sanchez et al., Int J Oral Maxillofac Implants, (2003), 18(1): 93-103. For purposes of supplementing cell culture media with these growth factors, platelet lysate was shown to be superior to both platelet adhesion and platelet aggregation methodologies. Doucet et al., J Cell Phys, (2005), 205: 228-236.
Despite enhancing cell growth, the use of unprocessed platelet lysate poses its own set of complications. Clinical uses of ex vivo cellular therapy have used FBS as a cell culture growth supplement until recently, and considerations regarding the use of platelet lysate in clinical applications remain to be addressed. These considerations include the presence of cellular debris, clotting factors, and cryoprecipitate as well as the immunosuppressive characteristics of cells cultured in the presence of platelet lysate. Furthermore, the use of unprocessed platelet lysate in cell culture results in the conversion of fibrinogen to fibrin. Generally, heparin is added directly to the cell culture to prevent this conversion. See, e.g., Schallmoser et al., Tissue Eng Part C Methods, (2008), 14(3): 185-96. In spite of this use of heparin, large fibrous strands may form within the media and attach to the MSC monolayer. This carries the risk of infusing patients with these fibrous strands, potentially resulting in micro or macro-occlusions. Thus, there is a need to find improved methods of reducing the generation of fibrous strands.
The zymogen Prothrombin is produced in the liver. It is enzymatically cleaved at two sites by Factor Xa, producing thrombin. Thrombin, in turn, mediates clotting by the conversion of fibrinogen to fibrin. Fibrin may cross-link with the transglutaminase Factor XIII, forming a clot. The fibrin may coalesce into fibrous strands that are hazardous if transferred into a patient, as described above. Heparin may be used to increase the affinity of anti-thrombin for both thrombin and Factor Xa, potentially disrupting the clotting cascade.